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. 2022 May;29(3):487-497.
doi: 10.1007/s12282-022-01330-8. Epub 2022 Jan 13.

Single HER2-positive tumor cells are detected in initially HER2-negative breast carcinomas using the DEPArray™-HER2-FISH workflow

Lisa Grüntkemeier  1 , Aditi Khurana  2 , Farideh Zamaniyan Bischoff  3 , Oliver Hoffmann  1 , Rainer Kimmig  1 , Mathew Moore  2 , Philip Cotter  2 , Sabine Kasimir-Bauer  4
Affiliations

Single HER2-positive tumor cells are detected in initially HER2-negative breast carcinomas using the DEPArray™-HER2-FISH workflow

Lisa Grüntkemeier et al. Breast Cancer. 2022 May.

Abstract

Background: In breast cancer (BC), overexpression of HER2 on the primary tumor (PT) is determined by immunohistochemistry (IHC) or fluorescence in situ hybridization (FISH) to stratify samples as negative, equivocal and positive to identify patients (pts) for anti-HER2 therapy. CAP/ASCO guidelines recommend FISH for analyzing HER2/neu (ERBB2) gene amplification and for resolving equivocal HER2 IHC results. However, pre-analytical and analytical aspects are often confounded by sample related limitations and tumor heterogeneity and HER2 expression may differ between the PT and circulating tumor cells (CTCs), the precursors of metastasis. We used a validation cohort of BC patients to establish a new DEPArray™-PT-HER2-FISH workflow for further application in a development cohort, characterized as PT-HER2-negative but CTC-HER2/neu-positive, to identify patients with PT-HER2 amplified cells not detected by routine pathology.

Methods: 50 µm FFPE tumor curls from the validation cohort (n = 49) and the development cohort (n = 25) underwent cutting, deparaffinization and antigen retrieval followed by dissociation into a single-cell suspension. After staining for cytokeratin, vimentin, DAPI and separation via DEPArray™, single cells were processed for HER2-FISH analysis to assess the number of chromosome 17 and HER2 loci signals for comparison, either with available IHC or conventional tissue section FISH. CTC-HER2/neu status was determined using the AdnaTest BreastCancer (QIAGEN, Hilden, Germany).

Results: Applying CAP/ASCO guidelines for HER2 evaluation of single PT cells, the comparison of routine pathology and DEPArray™-HER2-FISH analysis resulted in a concordance rate of 81.6% (40/49 pts) in the validation cohort and 84% (21/25 pts) in the development cohort, respectively. In the latter one, 4/25 patients had single HER2-positive tumor cells with 2/25 BC patients proven to be HER2-positive, despite being HER2-negative in routine pathology. The two other patients showed an equivocal HER2 status in the DEPArray™-HER2-FISH workflow but a negative result in routine pathology. Whereas all four patients with discordant HER2 results had already died, 17/21 patients with concordant HER2 results are still alive.

Conclusions: The DEPArray™ system allows pure tumor cell recovery for subsequent HER2/neu FISH analysis and is highly concordant with conventional pathology. For PT-HER2-negative patients, harboring HER2/neu-positive CTCs, this approach might allow caregivers to more effectively offer anti-HER2 treatment.

Keywords: Circulating tumor cells; DEPArray™; Early breast cancer; HER2; HER2/neu FISH; Tumor heterogeneity.

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Conflict of interest statement

During study performance, FB was employed at Menarini Silicon Biosystem, AK was an employee at RDx working on a contract for Menarini for which the prinicpal invetsogator was FB and LG was paid by Menarini. SKB was (is) a consultant for QIAGEN. Currently, OH is receiving consulting fees from Riemser, Roche, Amgen, Pfizer, Elsai, Hexal, MSD, Novartis and Daiichi Sankyo and RK from Tesaro, Astra-Zeneca and Medtronic, respectively.

Figures

Fig. 1
Fig. 1
Exemplarily DEPArray™-HER2-FISH image for one sample from the validation cohort. Image shows single channels and overlays of DAPI (blue)/CC17 (green)/ERBB2 (red) and were taken at 63 × magnification
Fig. 2
Fig. 2
DEPArray™-HER2-FISH images for six patients being PT-HER2-neg/CTC-HER2/neu-pos. Images show overlays of DAPI (blue)/CC17 (green)/ERBB2 (red) and were taken at 63 × magnification
See this image and copyright information in PMC

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